This invention relates to analytical methods and apparatus, more particularly to methods, apparatus and sensors for detection of a substance of interest in a fluid sample.
There are many types of standard tests or assays for detection of the presence and/or concentration of specific substances in fluids. Until recently many of these assays required development of different reagents and protocols for each substance to be detected. Examples of these approaches include various enzyme assay protocols in biochemistry laboratories and the Technicon SMAC machines, duPont Automated Clinical Analyzer, and Kodak Ektachem 700 machines in clinical chemistry laboratories.
In the last two decades a new type of diagnostic test or assay has gained increasing use--the antibody-based assay, or immunoassay. In immunoassays, an antibody may be used, for example, to probe for the presence of a particular antigen, hapten, or other molecule. Immunoassays have several potential advantages over previous assays:
(a) they are procedurally generalizable; that is, the same type of assay procedures and reagents can be used to detect most antigens no matter what the chemical properties of a particular antigen are.
(b) they are highly specific; that is, they can potentially distinguish well between structurally related compounds.
(c) they are potentially very sensitive.
Immunoassays are a specific type of a more general assay strategy, the ligand/antiligand assay. All ligand/antiligand assays are based on two premises: (1) that certain pairs of substances (the ligand and the antiligand) have a strong and specific affinity for each other, that is, they will tend to bind to each other, while binding little or not at all to other substances; and (2) that methods and apparatus can be developed that allow detection of ligand/antiligand binding interactions once complexes have formed. As used herein, ligand is defined as the substance to be detected, and antiligand the substance used to probe for the presence of the ligand. (In some ligand/antiligand assays, an additional, perhaps modified, ligand may be used that competes with the substance to be detected for binding sites on the antiligand.)
In many cases, detection of a ligand/antiligand complex is made possible by labelling one component of the complex in some way, to make the entire complex "visible" to an appropriate detecting instrument. For example, radioimmunoassay (RIA) uses a radioisotope as a label. See U.S. Pat. Nos. 3,555,143 and 3,646,346. Enzyme immunoassays (EIA) use an enzyme that can produce a detectable color under appropriate conditions. See U.S. Pat. Nos. 3,654,090, 3,791,932 and 3,850,752. Similarly, fluorescence immunoassays (FIA) use a fluorescent label.
Yet another ligand/antiligand assay that is becoming increasingly important is the nucleic acid hybridization assay, e.g., the DNA probe assay, which uses a "probe" strand of nucleic acid as an antiligand to test for the presence of a complementary DNA sequence. DNA probe assays, like immunoassays, often use radioactive labels, fluorescent labels or enzyme labels.
Recently, other ligand/antiligand assay techniques have been developed that use less standard tags. For example, particle counting immunoassay uses small latex spheres which scatter light in known ways when the spheres agglutinate due to antigen-antibody interaction. Both immunoassays and DNA probe assays have used luminescent labels as well.
Labels can greatly increase the sensitivity of ligand/antiligand assays by associating the presence of the labelled complex with a detectable signal. However, label-using assays also often have associated disadvantages. First of all, the labelled immunoreagents must be prepared, which may require time and expense. Also, several processing steps may be necessary during the assay, such as addition of label, incubation to allow reaction between components, washing away of excess label, transfer to a detecting instrument, and detection of the labelled complex. Because of their relative complexity, many of these assays may consume considerable time and require skilled technical help. Such assays also may be relatively hard to automate, except by means of expensive equipment. Further, these assays generally monitor the level of only one ligand at a time. Further still the labelled reagents may be unstable, inconvenient, or dangerous to handle, as with the isotopes I.sup.125 or p.sup.32 and carcinogenic enzyme substrates. Also, using the above and other currently available label-dependent assays, it is not possible to monitor the level of antigen continuously; thus in order to follow the level of an antigen or antibody over an extended time, samples must be withdrawn at intervals, treated with label, and tested.
A number of techniques are in commercial use or under development that seek to avoid some of the problems noted above by avoiding the use of labels or by modifying how lables are used. Many such techniques probe for the formation of ligand/antiligand complexes by optical means. For example, rate nephelometry, a type of immunoassay, measures changes in the angular distribution of scattered light as antigens and antibodies form aggregates under certain conditions. This method is not very sensitive, however, and requires testing of two dilutions of the sample.
Pregnancy testing, syphilis testing, and blood antigen testing, among others, are often done by visual inspection for formation of precipitated or agglutinated antigen/antibody complexes. Strictly speaking, however, the cells or particles often used in these tests to make aggregation visible could be called labels. In any case they have some of the disadvantages associated with labels noted above, such as the lack of potential for continuous measurement or for making simultaneous determinations of multiple ligands in the same sample. A number of other immunoassay systems use some form of optical detection without relying on precipitin formation, agglutination or standard labels. See U.S. Pat. Nos. 3,975,238, 4,054,646 and 4,321,057.
There are also several types of immunoassays based on electrical detection of ligand/antiligand complexes. These assays focus on overcoming one or more of the disadvantages noted above. One such method is the resistive pulse technique taught by U.S. Pat. No. 4,191,739, which makes a bulk conductance measurement. This technique is a modification of the Coulter counter approach, whereby a conducting fluid containing nonconducting particles is passed through a narrow constricted channel, whose overall (bulk) resistance is measured. The overall resistance increases whenever a nonconducting particle traverses the channel, and the size of the "pulse" produced is proportional to the particle's volume. Thus, if particles coated with antibody, for example, are exposed to antigen under appropriate conditions, they will aggregate, and the increased size and number of the aggregates can be related to the amount of antigen present. However, the sensitivity of this technique is limited by the presence of self-aggregates that form during the manufacture of the antibody-coated particles themselves. U.S. Pat. No. 4,191,739 seeks to eliminate this problem and hence increases the sensitivity of this technique by coating two particle preparations of different sizes with antibody, and counting only aggregates containing both large and small particles as indicative of the presence of antigen. However, the technique, as disclosed, requires an expensive apparatus. Also, it requires a label (the particles to which antibody or other antiligand is attached), and it cannot easily be adapted to measure multiple ligands at the same time in a single sample fluid, nor to measure a continuously varying sample.
U.S. Pat. Nos. 4,236,893 and 4,242,096 relate to the use of a piezoelectric oscillator that has been coated with antigen to detect the presence of antigen or antibody in a fluid sample. More specifically, there is a change in the frequency of the oscillator as its mass changes due to binding of antibody to the antigen on its surface. Although this assay does not require labelled reagents or sophisticated instrumentation, as disclosed it requires a complex protocol including removing the sensor from solution after exposure to the fluid sample and drying it before measurements can be made.
U.S. Pat. No. 4,0543,646 relates to the use of capacitance as one of several ways to measure the relative thickness of an antigen/antibody bimolecular layer. In particular, conducting substrate is exposed to an antigen, antibody, or other antiligand, whereby a monomolecular layer of antiligand forms on the conducting surface that will in general be an electrical insulator when dry. This surface is then exposed to a solution containing the ligand of interest, a layer of which is also an insulator when dry. Finally, a mercury drop or other electrode is contacted with the dry ligand layer. The conducting substrate and the mercury drop comprise the two conducting plates of a capacitor separated by an insulating layer whose thickness depends on the amount of ligand that has bound to the antiligand. The capacitance of this capacitor is then measured by means of a suitable instrument. This technique does not require labelled immunoreagents or expensive instrumentation. However, like the piezoelectric assay above, it requires that the ligand-containing capacitor be removed from solution and dried before measurements can be made.
Other types of electrical assays have sought to simplify ligand/antiligand assay procedures by measuring changes in electrical properties associated with a surface or interface in contact with an electrolyte. For example, when a surface such as a metal electrode is exposed to an electrolyte solution, strong local gradients of electrical charge and potential arise in the region of the electrode/electrolyte interface. Because the gradients and associated electrical potentials are strong, ligand molecules that interact with an immunoreagent or other antiligand immobilized at the interface can have a considerable effect on the overall electrical properties at the interface and thus can generate strong electrical signals of various kinds. Examples of assays based on the above phenomena include voltammetric assay as taught in U.S. Pat. No. 4,233,144; field effect transistor-based assays and other semiconductor-based assays as taught in U.S. Pat. Nos. 4,238,757 and 4,334,880; electrical reactance assays as taught in U.S. Pat. No. 4,219,335; antibody electrode assays (Analytical Chem. 56,801 (1984); Chem. & Eng. News, Apr. 2, 1984, p. 32) and other potentiometric assays, e.g., as taught in U.S. Pat. Nos. 4,151,049 and 4,081,334.
Such interface-based assays can be fast, simple to perform, and continuous, since a sample can be monitored as long as it is in contact with the interface. Such assays can also be label-independent and can be adapted to monitor multiple analytes simultaneously. However, interface electrical properties can be affected in many non-specific ways, such as by variations in pH or electrolyte composition or by non-specific adsorption of species in solution onto, or into, the surface or interface. Thus, methods based on measurement of such properties are often subject to interferences that are unpredictable or hard to control. Further, many of these methods require preparation of a membrane or molecular layer at or on the interface that contains ligand or antiligand, and this can be hard to do reproducibly.
It would therefore be advantageous to have a ligand/antiligand assay system which retains the advantages while overcoming the disadvantages associated with the above-described assays. More specifically, it would be advantageous to have an economical assay which is quick (can be completed in less than a minute), capable of being continuous, capable of detecting the presence of multiple ligands simultaneously in a fluid sample, simple to perform and label-independent. It would also be advantageous to have an apparatus which enables such an assay to be performed and which is miniaturizable in the sense that it allows measurement of ligand/antiligand interaction in an extremely small volume.